Submit a Manuscript: http://www.f6publishing.
com World J Gastroenterol 2017 September 7; 23(33): 6164-6171
DOI: 10.3748/wjg.v23.i33.6164
Retrospective Study
Ketogenic diet poses a significant effect on imbalanced
gut microbiota in infants with refractory epilepsy
Gan Xie, Qian Zhou, Chuang-Zhao Qiu, Wen-Kui Dai, He-Ping Wang, Yin-Hu Li, Jian-Xiang Liao, Xin-Guo
Lu, Su-Fang Lin, Jing-Hua Ye, Zhuo-Ya Ma, Wen-Jian Wang
Gan Xie, He-Ping Wang, Zhuo-Ya Ma, Wen-Jian Wang,
Department of Respiratory Medicine, Shenzhen Children’s
Hospital, Shenzhen 518026, Guangdong Province, China
Jian-Xiang Liao, Xin-Guo Lu, Su-Fang Lin, Jing-Hua Ye,
Department of Pediatric Neurology, Shenzhen Children’s
Hospital, Shenzhen 518026, Guangdong Province, China
Qian Zhou, Chuang-Zhao Qiu, Wen-Kui Dai, Yin-Hu Li,
WeHealthGene Institute, Shenzhen 518129, Guangdong
Province, China
Author contributions: Dai WK designed the study and Wang
WJ managed the project; Zhou Q and Qiu CZ interpreted the
data; Xie G and Zhou Q wrote the manuscript; Qiu CZ and Li
YH conducted bioinformatics analysis; Wang HP and Ye JH
collected sample information; Liao JX, Lu XG, Lin SF and Ma
ZY contributed to the study design and patients’ diagnoses; all
authors read and approved the final manuscript. Xie G, Zhou Q
and Qiu CZ contributed equally to this work.
Supported by the Innovation Fund of Science and Technology
Commission of Shenzhen Municipality, China, No. JCYJ-
2015-0403100317071.
Institutional review board statement: The study was
reviewed and approved by the Institutional Review Board of
Shenzhen Children’s Hospital.
Conflict-of-interest statement: The authors declare that they
have no competing interests.
Data sharing statement: Sequencing data are available from
the NCBI Sequence Read Archive (SRA) database (Accession
number: SRP100388).
Open-Access: This article is an open-access article which was
selected by an in-house editor and fully peer-reviewed by external
reviewers. It is distributed in accordance with the Creative
Commons Attribution Non Commercial (CC BY-NC 4.0) license,
which permits others to distribute, remix, adapt, build upon this
work non-commercially, and license their derivative works on
different terms, provided the original work is properly cited and
WJG|www.wjgnet.com
ISSN 1007-9327 (print) ISSN 2219-2840 (online)
ORIGINAL ARTICLE
the use is non-commercial. See: http://creativecommons.org/
licenses/by-nc/4.0/
Manuscript source: Unsolicited manuscript
Correspondence to: Wen -Jian Wang, Doctor, Director,
Department of Respiratory Medicine, Shenzhen Children’s
Hospital, No. 7019, Yitian Road, Shenzhen 5-18026, China.
dhbk2005-@163.com
Telephone: +86-755---83936101
Fax: +86-755---83009800
Received: April 24, 2017
Peer-review started: May 4, 2017
First decision: June 5, 2017
Revised: June 9, 2017
Accepted: July 12, 2017
Article in press: July 12, 2017
Published online: September 7, 2017
Abstract
AIM
To investigate whether patients with refractory
epilepsy and healthy infants differ in gut microbiota
(GM), and how ketogenic diet (KD) alters GM.
METHODS
A total of 14 epileptic and 30 healthy infants were
recruited and seizure frequencies were recorded. Stool
samples were collected for 16S rDNA sequencing using
the Illumina Miseq platform. The composition of GM in
each sample was analyzed with MOTHUR, and inter-
group comparison was conducted by R software.
RESULTS
After being on KD treatment for a week, 64% of epileptic
infants showed an obvious improvement, with a 50%
decrease in seizure frequency. GM structure in epileptic
6164 September 7, 2017|Volume 23|Issue 33|

infants (P1 group) differed dramatically from that in
healthy infants (Health group) . Proteobacteria, which
had accumulated significantly in the P1 group,
decreased dramatically after KD treatment (P2 group).
Cronobacter predominated in the P1 group and
remained at a low level both in the Health and P2
groups. Bacteroides increased significantly in the P2
group, in which Pre- votella and Bifidobacterium also
grew in numbers and kept increasing.
CONCLUSION
GM pattern in healthy infants differed dramatically
from that of the epileptic group. KD could significantly
modify symptoms of epilepsy and reshape the GM of
epileptic infants.
Key words: Ketogenic diet; Cronobacter; Seizures; Gut
microbiota; Epilepsy
© The Author(s) 2017. Published by Baishideng
Publishing Group Inc. All rights reserved.
Core tip: Many infants with epilepsy are refractory to
current antiepileptic drugs, and ketogenic diet (KD) could
help to moderate seizure frequency as an alternative
treatment. A large number of reports have demonstrated
that gut microbiota (GM) can affect children’s
neurodevelopment. Concurrently, GM could be
dramatically affected by diet. KD could rapidly alter GM
and alleviate seizure frequency in infants with refractory
epilepsy. The GM structure of epileptic infants
- comprising large numbers of pathogens, such as
Streptococcus - differed from that of healthy controls.
After KD therapy, GM of epileptic patients changed
significantly, with fewer pathogens and more beneficial
bacteria.
Xie G, Zhou Q, Qiu CZ, Dai WK, Wang HP, Li YH, Liao JX,
Lu XG, Lin SF, Ye JH, Ma ZY, Wang WJ. Ketogenic diet
poses a significant effect on imbalanced gut microbiota in
infants with refractory epilepsy. World J Gastroenterol 2017;
23(33): 6164-6171 Available from: URL: http://www.wjgnet.
com/1007-9327/full/v23/i33/6164.htm DOI: http://dx.doi.
org/10.3748/wjg.v23.i33.6164
INTRODUCTION
Pediatric epilepsy is widespread, with complications
including cognitive impairment, delayed neurodevelop-
ment and loss of bodily control[1,2]. Disequilibrium
between excitation and depression of the central
nervous system is acknowledged as the main factor in
epilepsy incidence[3]. Prior reports have identified
increased inflammatory reactions and pro-inflam-
matory cytokines, such as interleukin (IL)-6, IL-17 and
interferon, in the cerebrospinal fluid (CSF)[4]. Anti-
epileptic drugs (AEDs) and surgery are the main
conventional treatments for infants with epilepsy[5].
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Xie G et al . KD alters GM in epileptic infants
However, there are still 30% of epileptic infants who
suffered from therapeutic futility and recurrent attacks.
A growing number of reports indicated that KD is a
promising therapeutic alternative for infants with refractory
epilepsy, as it has been shown to ameliorate their clinical
symptoms, including the frequency of seizures[6-10]. It
remains unclear exactly how this occurs. Several reports
implicated changed neurotransmitters after KD therapy,
including γ-aminobutyric acid (GABA), monoamines and
glutamate[7,11]. Dahin et al[12] and Freeman et al[13] also
identified increased ketone bodies (KBs) and decreased
dopamine and serotonin[12,13]. However, Sariego-Jamardo et
al[14] found little change of neurotransmitters, pterins and
amino acids in the CSF of KD responders as opposed to non-
responders. These discrepant findings suggested a need for
the further elucidation of the mechanisms of KD therapy.
Several studies showed that diet posed a signifi- cant
effect on GM[8,15]. A high-fat diet induced selective
enrichment of bile-metabolizing microbiota, such as
Bacteroides[16], whilst high-fiber foods promoted the
accumulation of plant-polysaccharide fermenting
microbial organisms, including Prevotella and Clostri-
dium[16]. A number of reports implicated involvement of
GM in enteric nervous system, blood-brain barrier and
glial cell development, all of which were pivotal to
behavioral control and cognitive progression[17,18]. GM
could produce neurotransmitters and gut hormones
directly[19] or indirectly by producing signaling molecules
to regulate host cells[20]. GM-derived short-chain fatty
acids (SCFAs) could stimulate enterochromaffin cells to
produce serotonin[21]. Wikoff et al[22] also documented
decreased serotonin in peripheral serum in the absence
of GM. Moreover, Clostridium sporogenes and
Ruminococcus gnavus promoted decarboxylation of
tryptophan to tryptamine, which modulated mood and
appetite through amine-associated receptors[23]. Based
on the involvement of GM in the gut-brain axis,
increasing reports demonstrated imbalanced GM in
neurogenic diseases (NDs), including autism-spectrum
disorder, Parkinson’s disease, and depression[24].
However, GM dysbiosis in childhood epilepsy remains
unexplored.
Previous studies declared that short-term dietary
intake could rapidly alter human GM[8,15]. In this
study, we performed a comparison between diseased
infants (before and after KD treatment) and healthy
controls, to explore if and how GM of infants with
refractory epilepsy differed with that of age-matched
healthy subjects. We also evaluated the therapeutic
effect of KD on refractory epilepsy and the changes
in GM after treatment. It is hoped that this research
will help to bridge some gaps in the current
understanding of refractory childhood epilepsy.
MATERIALS AND METHODS
Sample collection
We enrolled 14 pediatric patients with refractory
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 Xie G et al . KD alters GM in epileptic infants

epilepsy (aged 1.95 ± 3.10 years, 11 male and 3 Shannon index

3.5
female) in Shenzhen Children’s Hospital, according to
the following inclusion criteria: Convulsion more than
four times per week after treatment with ≥ 3 AEDs; 3.0
no antibiotic exposure for at least 1 mo; no known
genetic metabolic disorders or severe systemic
2.5
illnesses; and successive KD therapy for at least 1
wk. KD was provided by Zeneca (Shenzhen, China),
including Qitong ketogenic liquid milk (3.4 g protein, 2.0

8.0 g lipid and 0.6 g carbohydrate per 100 g milk),

Qitong ketogenic cookies and Qitong ketogenic set- 1.5
meal pack- ages[25].
Healthy subjects (aged up to 3 years, 15 male
1.0
and 15 female) were also recruited based on the
following criteria: No antibiotic exposure for at
least 1 mo before this study, no disease P1 P2

symptoms for at least 1 mo following recruitment, Health

and no history of seizures (Supplementary Table
1). Fisher’s exact test was used to evaluate the
effect of gender and age on GM composition.
Figure 1 Gut microbial diversity of the three groups. Distribution of
Shannon index (evenness) is shown. Red, blue, and green represent the
Health, P1 and P2 groups, respectively. The gut microbiota (GM) of the healthy
infants was more stable than that of the other two groups.

DNA extraction, library construction and sequencing

The genomic DNA of microbiota was extracted from to 305190). The number of assembled tags
stool samples using the Power Soil DNA Isolation Kit averaged 22800, with a range from 12655 to
(Mo Bio Laboratories, Carlsbad) following the 27337. Both gender and age had no significant
manufacture’s protocol. The hypervariable V3-V4 effect on GM (P = 0.069 and 0.234, respectively).
region of the 16S rRNA gene was amplified using PCR
kit (TransGenAP221-02, Peking), and DNA products GM of healthy individuals differs dramatically with
were quantified by gel electrophoresis and Qubit that of diseased infants
(Thermo Fisher, Singapore). After library Shannon index analysis indicated higher GM diversity in
construction, the qualified libraries were sequenced healthy infants, in comparison with infants with
using the Illumina MiSeq Sequencing platform refractory epilepsy (Figure 1, Supplementary Table 2).
(Illumina, San Diego). PCA of GM profile also identified that healthy infants
could be clearly distinguished from patients (Figure 2,
Taxonomy classification and diversity detection Supplementary Table 3). The phylum Firmicutes
After filtration, overlapped paired reads were asse- predominated in patients (45.82%) and was un-
mbled as tags with FLASH (v1.2.11), and clustered to changed after KD therapy (47.00%) (Supplementary
operational taxonomic units (OTUs) through USEARCH Table 4). Bacteroidetes accounted for 53.01% of GM in
(v7.0.1090)[26]. Representative OTUs were mapped healthy infants, followed by Firmicutes (34.38%). After
against the Greengenes database (v201305)[27] and KD treatment, Bacteroidetes increased from 26.75% to
classified with RDP classifier (v2.2)[28]. The diversity of 38.71%. Actinobacteria was enriched in healthy infants
(8.49%) and occupied a lower percent in patients
microbiota was calculated with MOTHUR (v1.31.2)[29].
(2.38% before treatment and 2.92% after treatment).
Principal component analysis and statistical analysis Proteobacteria was highly accumulated in infants with
PCA was performed with R software (v3.2.5). Wilco- refractory epilepsy (24.34%) and decreased dramatically
xon rank-sum test was used to compare GM in dis- after KD therapy (10.77%). At the genus level,
eased infants and healthy controls (Health group). Cronobacter was dominant in the patients (23.30% vs
Comparative analysis between the epileptic infants 0.00% in the healthy group). By contrast, healthy
before (P1 group) and after treatment (P2 group) subjects harbored more than twice Bacteroides
was conducted by Wilcoxon signed-rank test. Linear (42.68%) than infants with refractory epilepsy
discriminant analysis Effect Size (LEfSe) analysis was (17.93%). Prevotella and Bifidobacterium also
used to identify microbial species which were accumulated in the healthy group (7.25% and 7.84%,
apparently enriched in a specific group. respectively) (Supplementary Table 5).

KD therapy ameliorates epilepsy and GM of

RESULTS patients started to improve
Data output and patients’ characteristics After a week of KD therapy, 3 (21%) patients were
The average number of high-quality sequencing reads seizure-free and 6 (43%) had a 50% to 90% decrease of
produced for each sample was 117196 (range, 31900 seizure frequency (Supplementary Table 1). The

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 Xie G et al . KD alters GM in epileptic infants

6
Health
P1
P1

2
PC2 (8.23%)

-2

-4

-6

-6 -4 -2 0 2 4 6

PC1 (26.95%)

Figure 2 Principal component analysis. Each plot in the principal component analysis (PCA) graph stands for a sample. Red, blue and green colors represent the
Health, P1 and P2 groups, respectively.

P1
Roseburia

Erysipelatoclostridium

Acidaminococcus
Bacteroides
Parabacteroides
Collinsella
Alistipes
Anearostupes

Dysgonomonas Streptococcus

Faeclibacterium Health

Blautia
Haemophilus
Ruminococcus
Prevotella
Bifidobacterium

Gemmiger

Cronobacter
P2

Figure 3 Gut microbiota structures in the Health, P1 and P2 groups at the genus level. SVG package (version 1.1) was used to produce the paragraph. The size
of the circle representing each genus was determined by the relative abundance of the three groups, and the width of line linking the P1, P2 and Health groups
indicates the relative abundance of each group.

remaining 5 (36%) infants experienced no significant of the Health group, by comparison with P1 group
improvement in seizure control (Supplementary Table (Figures 3 and 4). After KD treatment, Bacteroides
1). GM of the P2 group was more similar to that increased significantly, by 24.42%. Prevotella also

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 Xie G et al . KD alters GM in epileptic infants

Health P1 P2

Odoribacter
Eggerthellales
Eggerthellaceae
Eggerthella
Peptoclostridium
Bacillales
Parvimonas
Bacillaceae
Bacillus
Solobacterium
Brochothrix
Listeriaceae
Cronobacter
Enterobacteriales
Enterobacteriaceae
Gammaproteobacteria
Erysipelotrichaceae
Erysipelotrichales
Erysipelotrichia
Erysipelatoclostridium
Bacilli
Ruminiclostridium
Lactobacillales
Streptococcaceae
Streptococcus
Hungdatella
Lachnoclostridium
Clostridiaceae
Enterococcus
Enterococcaceae
Klebsiella
Butyricicoccus
Myroides
Peptostreptococcus
Mogibacterium
Weissella
Psychrobacter
Peptoniphilus
Clostridiales_incertae_sedis_XIII
Leuconostocaceae
Lacticoccus
Sphingomonadaceae
Acinetobacter
Bacteroidaceae
Bacteroides
Actinobacteria
Bifidobacteriaceae
Bifidobacteriales
Bifidobacterium
Faecalibacterium
Actinobacteria
Ruminococcus
Betaproteobacteria
Pasteurellales
Pasteurellaceae
Haemophilus
Burkholderiales
Escherichia
Sutterellaceae
Clostridium
Actinomycetales
Sutterella
Staphylococcus
Candidatus_saccharibacteria
Staphylococcaceae
Campylobacter
Epsilonproteobacteria
Campylopacterales
Campylobacteraceae
Actinmycetaceae
Actinomyces
0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 4.5
LDA score (log10)

Figure 4 Significantly enriched gut microbiota components in the Health, P1 and P2 groups. LEfSe analysis was applied to detect the gut microbiota (GM)
components in the three groups. Red, green, and blue represent the Health, P1 and P2 groups, respectively. The LDA score was set as ≤ 2. The enrichment degree
is proportional to the LDA score.

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increased in the P1 group from 0.37% to 1.85% after
KD treatment (Figure 3 and Supplementary Table 5).
Cronobacter decreased sharply in after-treatment
patients, from 23.3% to 10.44 % (Figures 3 and 4
and Supplementary Table 5). KD exposure also
induced a decrease in Erysipelatoclostridium (by
8.67% in the P1 group and 4.89% in the P2 group);
it represented just 0.64% in healthy infants (Figures
3 and 4 and Supplementary Table 5). Streptococcus,
Alistipes, Ruminiclostridium, Barnesiella and
Enterococcus also decreased after KD therapy
(Figures 3 and 4 and Supplementary Table 5).
DISCUSSION
KD is increasingly used for the treatment of refractory
epilepsy in childhood, but the mechanism remains
unclear. Previous reports indicated that GM played an
important role in the gut-brain axis[24], and was affected
significantly by intake of high-fat food[16]. This study
focused on differed GM structures between healthy and
epileptic infants, as well as altered GM in patients after
one week of KD treatment. The results pointed to an
imbalanced GM in patients and a significant
improvement after KD therapy.
Proteobacteria comprises a variety of notorious
pathogens, such as Escherichia, Salmonella and
Vibrio. It accounted for 24.34% in pediatric patients
and decreased dramatically after KD treatment.
Bacteroidetes was dominant in healthy infants and
increased largely in after-treatment patients.
We identified accumulated Bacteroides in healthy
subjects as well as in patients after treatment.
Bacteroides was reported to digest and metabolize high-
fat food and to regulate the secretion of IL-6 and IL-17
in dendritic cells (DCs), a process strongly associated
with seizure severity of epileptic patients[4,16]. However,
patients-enriched Cronobacter decreased dramatically
after KD therapy. Prior reports demonstrated that there
were multiple virulence determinants of Cronobacter,
including Cronobacter plasminogen activator and ferric
ion transporter protein, which paly a detrimental role in
human health[30-32]. Prevotella is a robust producer of
SCFAs[33], which could protect the intestinal mucosa and
function as neurotransmitters. Previous reports also
indicated that SCFAs mediated nervous impulse and
mitigated Parkinson’s disease[33,34]. Similarly,
identified increased Prevotella in the Health and P2
group, when compared with the P1 group. Some other
genera also offer clues to epilepsy recovery, such as
Erysipelatoclostridium, Blautia, Bifidobacterium and
Streptococcus. Bifidobacterium was well known to be
beneficial to health[35], and Streptococcus, a common
pathogen, played a role especially in respiratory
diseases[36]. Although GM imbalance in diseased infants
was identified and GM improved after KD treatment,
more exploration was needed to elucidate the contri-
bution of a healthy GM to epilepsy onset/recovery.
This study revealed that KD can mitigate the sym-
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Xie G et al . KD alters GM in epileptic infants
ptoms of epilepsy and correct an imbalanced GM
in epileptic infants. However, further analysis is
needed to unravel how GM may be involved in
epilepsy onset/ recovery.
There are some limitations that need to be clarified.
First, 16S rDNA analysis identified microbes at the genus
level, which makes it difficult to unravel different
microbes at the species or function level. Second, it
would be more useful to evaluate the efficacy of KD
treatment and its effect on the GM if this could be done
with a longer period of follow-up. Third, an animal model
might be applied to demonstrate whether GM imbalance
could induce epilepsy associated symptoms. Considering
these limitations, we are planning to perform
metagenomic analysis on GM of healthy and epileptic
infants. This will provide more insights into distinct
metabolic networks in imbalanced GM.
In conclusion, we found that GM of infants with
refractory epilepsy differed dramatically from that of
healthy infants. Epileptic patients harbored significantly
enriched pathogens and decreased beneficial bacteria.
Although this study provides new insight into the
involvement of GM in pediatric refractory epilepsy, the
gap between KD and epilepsy recovery is still huge. To
uncover the mechanism and pathogens involved in
refractory infantile epilepsy, further research should
underscore functional gene networks in GM.
ACKNOWLEDGMENTS
We thank the staff of WeHealthGene who
contributed to the project, but whose names are
not included in the author list.
COMMENTSCOMMENTS
Background
Infants with refractory epilepsy could not be cured by several anti-epileptic
drugs (AEDs) and ketogenic diet (KD) was increasingly used as an alternative
therapy to refractory epilepsy. High-fat diet was reported to pose a significant
impact on gut microbiota (GM), which could regulate neural systems.
Research frontiers
Previous reports demonstrated that GM could affect neural systems by
secreting metabolites as neurotransmitters. In parallel, the gut -brain axis is a
research hot spot in biomedicine, including the study of autism, Parkinson’s
disease, and depression.
we
Innovations and breakthroughs
This study showed that the GM pattern of diseased infants differs significantly
from that of healthy controls. The decreased number of dominant pathogens
and significantly increased number of beneficial bacteria after KD treatment
offer new insight into KD therapy for epilepsy.
Applications
This study found several types of bacteria altered in the GM, suggesting that
these bacteria could be monitored as biomarkers to provide an important
reference for epilepsy treatment.
Terminology
GM, which consists of many kinds of bacteria including pathogens, commensals,
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 Xie G et al . KD alters GM in epileptic infants
and probiotics, plays an important role in the human body.
Peer-review
The authors have performed important research in pediatric epilepsy. They
discovered that the composition of the GM in healthy and diseased infants was
significantly different, specifically in healthy infants as opposed to those with
refractory epilepsy. Bacterial patterns were dramatically changed after KD
therapy, and this was associated with a reduction in the frequency of seizures.
These findings should enhance our knowledge of the relationship between
epilepsy and GM and provide new insight into the clinical treatment of epilepsy.
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P- Reviewer: Daniel F, Prakash N S- Editor: Qi Y
L- Editor: Wang TQ E- Editor: Xu XR
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